The present invention relates to methods and an apparatus for extracting and purifying taxol and derivatives thereof from source materials containing the same. In particular, the methods and apparatus can be used in separating taxol and its derivatives from needles and other parts of yew trees.
Numerous compounds that exhibit therapeutic activity in the treatment of diseases have been isolated and identified in organic solvent extracts from plant material, microorganisms and marine organisms. Some of the compounds that have been demonstrated to have a particularly good physiological activity have been used as chemotherapeutic or anti-HIV agents. However, investigation into the therapeutic utility of these compounds has been hindered by the extreme abundance of natural sources to be screened and the inefficiencies of conventional extraction techniques.
Examples of such useful compounds include plant alkaloid derived from Vinca rosea and its semisynthetics vinblastine and vincristine, and taxol (NSC 125973), a diterpene alkaloid plant product derived from the pacific yew, Taxus brevifolia. 
Taxol
Taxol is one of a class of drugs inhibiting mitosis of eukaryotic organisms. It promotes polymerization of tubulin and stabilizes the structure of intracellular microtubules to produce an anti-cancer effect (see, Schiff, P. B. et al., Nature 277, 665-667 (1979)).
In the 1960""s, taxol was discovered as a result of a plant screening program organized by the US National Cancer Institute (NCI). After cytotoxic activity was first found in a crude extract from the bark of the Pacific yew tree, Taxus brevifolia, the active compound was isolated in 1966. It was named taxol in 1967, and its molecular structure was characterized by Wani et al. in 1971 (Wani, M. C. et al., J. Am. Chem. Soc., 93: 2325, 1971). Horwitzi et al. (1979) elucidated its active mechanism (Horwitz, F. B., et al., Nature, 1979, 277, 665 and Eric K. Rowinsky et al., J. National Can. Inst., 82, 11247 (1990)). According to NCI""s reports, the response rate to taxol of ovarian cancer, breast cancer or lung cancer patients, all who have exhibited no response to conventional anti-cancer drugs, were 30%, 50% and 20%, respectively (David, G., et al., J. Nat. Prod., 53, 1 (1990)).
Marketing approval of taxol as an anti-cancer drug started from 1992, when the US Food and Drug Agency (FDA) and the HPB (Canada) approved the use of taxol for the treatment of ovarian cancer. In 1994, the use of taxol for the treatment of breast cancer and Kaposi""s sarcoma was approved by the FDA. Subsequently, the FDA approved taxol for treating non-small-cell lung cancer (NSCLC) in 1997 and expanded the use of taxol to early breast cancer in 1999. In addition, taxol has been reported in studies to exhibit excellent effects on other types of cancer (Spencer, C. M. and Faulds, D., Drug. 48, 794-847 (1994)).
Conventional Preparation of Taxol
The conventional purification method is performed in four major steps. First, the acetone:hexane mixture from the extraction process is chromatographed on Florisil columns in a 70/30 hexane:acetone mixture to separate the taxol containing fractions. The taxol fractions are then concentrated to dryness. This step may be repeated as many as nine times. Second, taxol concentrates are crystallized from a methanol:water mixture and then recrystallized from an acetone:hexane mixture yielding 85 to 95% pure taxol. Third, the taxol is chromatographed on silica gel packed with either 2.5% isopropanol or 2.5% n-butanol in methylene chloride to yield approximately 98% pure taxol. Fourth, the taxol is dissolved in acetone, the solution filtered, and taxol recrystallized from an acetone:hexane mixture. This organic phase extraction and chromatographic purification process yields 99% pure taxol, which is about 0.014% of the milled bark (J. Liquid Chromatography, 12(11): 2117-2132 (1989); WO 92/07842).
The production of taxol by this technique is encumbered by the following: (i) time consuming extraction and purification procedures; (ii) long residence times in a harsh environment; and (iii) low overall yields. Also, the bark of T. brevifolia is usually obtained from mature trees (100 to 200 years old). The bark is thus in limited supply.
Many studies have been conducted to overcome such problems and to provide alternate routes for obtaining taxol. Some of the alternate routes include total chemical synthesis and cell culturing synthesis. Several total chemical synthesis methods were reported in 1994. However, these methods were difficult to commercialize due to their complicacy. This problem originated from the fact that taxol has several asymmetric carbon atoms and a complex structure. The production of taxol through cell culturing has also undergone some difficulties in commercializing because of the slow rate of cell growth and the easy browning of cells (U.S. Pat. No. 5,019,504, issued May 28, 1991).
Yew trees can be classified into the following genera: Amentotaxus, Austrotaxus, Cephalotaxus, Pseudotaxus, Taxus, Torreya, etc. Among them, Taxus genus includes, for examples, T. brevifolia, T. baccata, T. media, T. wallichiana, T. Canadensis, T. cuspidate, etc, which are generally considered to be suitable sources for extraction of taxol and its derivatives.
Yew trees, such as T. cuspidate, which have a high taxol content in their needles, have been planted plentifully with regard to landscape and are easy to cultivate in farm fields. However, the trees have high levels of wax and non-polar components, which makes the separation of taxol therefrom difficult. The total amount of taxol in organic solvent extracts from the bark was 25%, but the content of impurities was very high in the range of 35-42%. In order to easily separate taxol from T. cuspidate, one must first develop selective removal techniques of materials such as waxes and chlorophyll.
The term xe2x80x9cimpuritiesxe2x80x9d herein refers to all components of extracts from plants with the exception of taxol and its derivatives.
The use of supercritical fluid in the extraction of taxol has been proposed for improved extraction. The term xe2x80x9csupercritical fluidxe2x80x9d refers to a fluid that is above its critical pressure and above its critical temperature. A supercritical fluid has both the gaseous property of being able to penetrate anything and the liquid property of being able to dissolve materials into their components. In addition, it offers the advantage of being able to change density to a great extent in a continuous manner and by adjustment of the system pressure and temperature. This advantage is connected to simple control of its dissolving capability. As such, use of supercritical fluids is offered as a substitute for organic solvents in the fields of food industry and medical supplies.
Furthermore, supercritical fluids do not extract chlorophyll (M. T. Tena et. al. Analytical Chemistry, Vol. 69, No. 3, 521 (1997)). Particularly, supercritical carbon dioxide is advantageous in extracting desired ingredients from plants without chlorophyll extraction.
KR patent application Nos.1994-7829, 1994-36099, 1995-703845, 1996-19486 and 1996-0055302 describe methods for extracting taxol from plants with supercritical carbon dioxide.
In particular, KR patent application No.1994-7829 discloses a method and an apparatus for continuously separating and purifying taxol with high purity wherein supercritical fluid extraction is accomplished in a countercurrent manner. Cosolvents are also employed in the invention.
KR patent application No.1994-36099 discloses the use of n-hexane in removing non-polar substances prior to supercritical carbon dioxide extraction. Namely, it suggests a combining method of organic solvent extraction with supercritical carbon dioxide extraction.
KR patent application No.1995-703845 is a method for extracting taxol from the needles of ornamental yew comprising the steps: (a) dewaxing the needles by subjecting said needles to supercritical fluid, (b) subjecting said dewaxed needles to supercritical fluid and cosolvents and (c) separating taxol through column chromatography. However, this method has some problems, such as a risk of taxol loss during the dewaxing step and the incomplete extracting capability of the cosolvents employed.
KR patent application Nos. 1996-55302 and 1996-19486 disclose extraction methods similar to the methods indicated above.
Consequently, conventional supercritical fluid extraction techniques are not optimal for taxol extraction.
Taxol is linked to cell walls in plants through a weak chemical bond. Depending on the properties of solvents used, taxol can be partially extracted from plants. Supercritical carbon dioxide makes plants swell, which allows for the desired substances to be easily extracted, whereas its polarity makes the extraction of high polar and chemically linked substances (e.g., taxol) difficult (M. D. Luque de Castro et. al. Analytical Supercritical Fluid Extraction, Springer-Verlag, Berlin, p. 188 (1994), Y. Kim et. al. J. Chromatographic Science, Vol. 37, 457 (1999)).
Also, organic solvents cannot easily penetrate plants and are limited in their ability to extract taxol.
The present invention provides a novel method for preparing taxol and derivatives thereof, comprising a supercritical fluid extraction step, an organic solvent extraction step, and a chromatographic step (e.g., a multi-column chromatographic step). The invention overcomes limits in conventional supercritical fluid extraction by employing cosolvents optimized for taxol extraction in the first step.
In one aspect, the invention features a method for isolating Taxol or derivatives thereof from a source material. The method includes (a) extracting the source material with a supercritical fluid (e.g., supercritical carbon dioxide) and a cosolvent to obtain an extract; (b) liquidxe2x80x94liquid separating the extract with an organic solvent to obtain a solvent layer; and c) isolating Taxol or the derivatives thereof from the solvent layer by column chromatography. The steps can be conducted in a continuous or non-continuous manner. The ratio of the supercritical fluid to the cosolvent can be between 75:25 and 85:15.
A cosolvent serves to release taxol and its derivatives from the matrix of source materials. Preferably, the cosolvent has a xe2x80x94OH group in its chemical structure. For example, the cosolvent can be a mixture of water and at least one alcohol (e.g., methanol or ethanol). Examples demonstrate cosolvents that include water and alcohol, such as methanol and ethanol. At least one of the cosolvents can be used simultaneously. Preferably, a mixture of water and alcohol is used. A volume ratio of water to alcohol is preferably from 30:70 to 5:95 and more preferably 20:80. Further, additional solvents, such as acetic acid and triethylamine, can be added to the cosolvents, for example, to 1%(v/v). Acetic acid acidifies the supercritical fluid and cosolvents, and triethylamine bacifies the supercritical fluid and cosolvents. Cosolvent selection criteria are further described in Example 1 below. The volumetric ratio of water to at least one alcohol can be between 30:70 and 5:95 (e.g., 20:80).
In step (a) in accordance with the present invention, preferable conditions of the supercritical fluid comprise a temperature of 60 to 100xc2x0 C. and a pressure of 300 to 400 bars. More preferably, the temperature is in the range of 75 to 85xc2x0 C. and the pressure is in the range of 330 to 370 bars. Most preferably, about 80xc2x0 C. and about 350 bars are selected. A flow rate of supercritical fluids is preferably between 30 and 50 kg/hr. The supercritical fluid extracting step also can be performed at a temperature of 75 to 85xc2x0 C., a pressure of 330 to 370 bars, and a flow rate of 30-50 kg/hr of the supercritical fluid.
In step (b), preferable examples of the organic solvent include n-hexane. N-hexane can combine with the cosolvent (preferably, alcohol) from step (a) and then participate in liquidxe2x80x94liquid separation together with alcohol. N-hexane is preferably fed in an amount similar to that of the cosolvent. Namely, a volume ratio of n-hexane to the cosolvent (preferably alcohol) becomes 1:1. Step (b) may be repeated one to three times. The extracting and liquidxe2x80x94liquid separating steps can be performed continuously or non-continuously.
Step (c) can include multi-column chromatographic procedures. Each column chromatographic procedure can be performed continuously or non-continuously.
Examples of resins that can be used include silica gel, RP-18, Sephadex, and pentafluorophenyl resin. Each of the multiple columns includes a column resin. For example, silica gel, RP-18, and Sephadex column resins can be used. A column including a pentafluorophenyl resin can be used as the final column when multiple columns are used.
In silica gel column chromatography, silica gel may be used in an amount of 10 times the extract weight and a stepwise gradient system of chloroform:methanol (e.g., 1:0xe2x86x920:1 v/v) can be used as an eluant. Preferably, fractions containing taxol and its derivatives can be obtained. Alternatively or additionally, silica gel may be used in an amount of 33 times the weight of extracts applied to the column, and elution may be performed with dichloromethane:methanol (e.g., 85:15 v/v).
The amount of the RP-18 resin can be 10 times the extract weight applied to the column, and methanol can be used as eluant. Fractions containing Baccatin III can be eluted from the RP-18 resin.
The amount of the Sephadex resin can be 65 times the extract weight applied to the column, and methanol can be used as eluant. Cephalomannine can be eluted from the Sephadex resin.
In Sephadex column chromatography, Sephadex resin may be used in an amount of 65 times the extract weight and methanol can be employed as an eluant. Preferably, fractions containing cephalomannine may be obtained by the elution.
In one embodiment of the invention, a final column used in step (c) may include a pentafluorophenyl resin. The amount of the pentafluorophenyl resin can be 133 times the extract weight applied to the column. An acetonitrile:water gradient system can be used as eluant. Acetonitrile content can be changed from 60% to 90% during the course of the chromatography (e.g., changed from 60% to 80% during the first 40 minutes and then a further increase to 90% during an additional 10 minutes).
The method further can include recrystallizing the isolated taxol and derivatives thereof (e.g., to increase purity of the product). A solution of water:methanol (1:1 v/v) can be used.
The source material to which the method of the invention can be applied is plant material containing taxol or its derivatives. The most suitable plants are species of Taxus, such as T. brevifolia, T. baccata, T. media, T. wallichiana and T. canadensis, T. cuspidate, but are not limited thereto. Among the Taxus species, T. cuspidate is particularly preferred. Also, taxol and its derivatives can be extracted from the whole plant or from separated parts such as wood, stems, bark, roots, leaves (needles), seeds or mixtures thereof. The material can be dried. Preferably, the bark or the needles are used.
The invention also features an apparatus for isolating Taxol and derivatives thereof from source materials. The apparatus includes a reservoir for supercritical fluid, an extractor into which the source material can be fed, a separating vessel that separates the supercritical fluid from the extract mixtures, a liquidxe2x80x94liquid separator, and at least one column chromatography, all being placed online (e.g., all within a single production line). The apparatus further can include at least one separating vessel, which separates the supercritical fluid from the extract mixtures in a continuous form. The apparatus can allow the recovery and recycling of a full amount of solvents used in the liquidxe2x80x94liquid separator with the exception of solvents transferred into column chromatography.
In one preferable embodiment of the invention, the apparatus comprises at least one separating vessel, which separates the supercritical fluid from the extract mixture, in a continuous form.
In another preferable embodiment of the invention, the apparatus allows the recovery and recycling of a full amount of solvents used in the liquidxe2x80x94liquid separator with the exception of solvents transferred into column chromatography. One preferable embodiment of such apparatus is illustrated in FIG. 1, which exemplifies the use of a n-hexane and alcohol mixture in the liquidxe2x80x94liquid separator.
The following advantageous effects were obtained by the present invention:
The invention can provide a simple method for extracting and separating taxol and its derivatives by combining a supercritical fluid extraction with an organic solvent separation and avoid the complicated extraction procedure of conventional taxol extraction methods.
The invention can provide certain optimal cosolvents for taxol extraction in view of both, (i) the capability of dissolving taxol and its derivatives, and (ii) the ability of releasing taxol and its derivatives from the matrix of source materials. As a result, the invention provides an improved taxol extraction in terms of efficiency.
The invention also provides special conditions between the supercritical fluid and cosolvents for improved taxol extraction. Further, the invention illustrates a cellulose paper-taxol model showing the cosolvents"" mechanism of action during the extraction. The inventors deduced from the model that water may be useful as a cosolvent and supported the conclusion by providing experimental results to show that a cosolvent mixture of water and alcohol increases the extraction yield (see Examples hereinafter).
The method of the invention increased the yield of taxol to an amount 2 times that of conventional methanol extractions. An xe2x80x9cextraction selectivityxe2x80x9d that is related to the absolute amount of taxol contained in extracts was demonstrated to increase 5.5 times as compared with the methanol extractions.
The invention also provides a chromatographic assembly comprising 5 columns, which allows a simple purification in comparison with conventional methods. Also, all of the resins used are confirmed to be recyclable.